1. Field of the Invention
The present invention relates to DNA sequences which encode an amino acid sequence homologous to a segment of Trichinella spiralis 53 kilodalton (kD) excretory-secretory antigen, recombinant polynucleotide molecules containing the sequences, and transfer and replication of the sequences in a transformed host to produce antigens useful as immunodiagnostic reagents or vaccines specific for T. spiralis
2. Description of the Art
Trichinellosis (known historically as trichinosis), caused by the nematode parasite Trichinella spiralis, is an important zoonotic disease of world wide distribution. Trichinellosis results from the ingestion of raw or undercooked meat, generally pork, containing the infective larval stage of the parasite. An important component of a program to control or eradicate trichinellosis is a specific and sensitive diagnostic test. A variety of direct and indirect tests have been used for the diagnosis of trichinellosis in swine and other species, the most recent of which is the enzyme-linked immunosorbent assay (ELISA). Initially, ELISA tests were performed with crude extracts of T. spiralis is muscle larvae as antigen and resulted in a high number of false-positive reactions, apparently due to cross-reactions with other parasite infections. Subsequent replacement of the crude antigen preparation with biochemically purified stichocyte antigens (G. L. Seawright et al., American Journal of Tropical Med. Hyg. 32: 1275-1284, 1983), culture-derived excretory-secretory (ES) antigens (H. R. Gamble et al., Veterinary Parasitology 13: 349-361, 1983), or antibody affinity-purified ES antigens (H. R. Gamble and C. E. Graham, American Journal of Veterinary Research 45: 67-74, 1984) resulted in near elimination of false-positive reactions; however, inconsistencies in the yield and purity of antigen obtained by these procedures has remained a problem.
Use of naturally derived antigens to T. spiralis has several disadvantages. It requires continual passage of the parasites in laboratory rodents. The rodents must be sacrificed 30-40 days post-infection at which time the parasites are collected for antigen isolation, and additional rodents are infected for subsequent isolations. Thus, production of naturally derived antigens is both time consuming and costly. Further, both the yield and purity of material can vary significantly between preparations. What is needed is an economical way to provide an unlimited source of T. spiralis antigens having reliable and reproducible purity.
Limited information is available about T. spiralis antigens. T. spiralis muscle larvae have been shown to excrete and secrete antigens in culture which are useful as immnodiagnostic reagents (Gamble et al., 1983, supra). Immunodominant antigens in ES have been identified in the 45,000 to 53,000 molecular weight range. Similar molecular weight antigens have been extracted from T. spiralis stichocyte cells using biochemical procedures (D. S. Silberstein and D. D. Despommier, The Journal of Immunology 132:898-904, 1984). Monoclonal antibody generated from spleen cells of mice infected with T. spiralis recognized an antigenic determinant unique to T. spiralis present on proteins of molecular weights 45,000, 49,000, and 53,000 (Gamble and Graham, 1984, supra; H. R. Gamble, Experimental Parasitology 59: 398-404, 1985, and U.S. Pat. No. 4,670,384). Two of the proteins (49,000 and 53,000 molecular weight) possessing this determinant were isolated together by monoclonal antibody-affinity chromatography, and the affinity-isolated antigen used successfully in an ELISA for swine trichinellosis (Gamble and Graham, 1984, supra). T. spiralis antigens have been reported to be glycoproteins (Gamble, 1985, supra, and D. S. Silberstein, Dissertation Abstracts International 45: 824B, 1984). A 48 kD antigen, presumed homologous to the 45 kD antigen of Gamble and Graham, 1984, supra, has been partially characterized. However, no function for the proteins has been elucidated and biological activity has yet to be assigned. Further, no data has been made available indicating which stage of development the corresponding messenger RNA which codes for the antigen is produced.
An attempt to prepare T. spiralis diagnostic antigens by recombinant DNA techniques has been reported (Abstract, D. S. Zarlenga and H. R. Gamble, Federation Proceedings 46: 2038, 1987). The researchers prepared cDNA clones using poly A mRNA isolated from T. spiralis muscle stage larvae; however, the recombinant antigens were unable to detect antibodies to T. spiralis using sera from experimentally infected animals (D. S. Zarlenga and H. R. Gamble, unpublished data). Further, none of the genes coding for antigens described in the Abstract were shown to code for any of the ES products of T. spiralis e.g., the 45, 49, and 53 kD immunodiagnostic antigens, (D. S. Zarlenga and H. R. Gamble, unpublished data).